Staphylococcus aureus is a virulent pathogen which is associated with a broad-spectrum of clinical infections. Its ability to colonize host tissues, and to persist and proliferate within host tissues requires the organism to circumvent innate host defense mechanisms. We have discovered that mammalian platelets store and secrete a family of antimicrobial peptides at potential sites of endovascular damage and microbial colonization that serve to both growth inhibit and kill S. aureus. In the previous grant period, we have delineated that the principal antimicrobial peptide which is secreted from platelets (thrombin-induced platelet microbicidal protein-1 [tPMP-1]), interacts with S. aureus in vitro by initial attachment to the cytoplasmic membrane, after which a microbicidal cascade is triggered in strains intrinsically susceptible to this peptide. In contrast, those strains which were engineered to be resistant to tPMP-1 in vitro (e.g., by transposon mutagenesis) do so by changing the basic biology of their cytoplasmic membrane target for tPMP-1. In vitro susceptibility to tPMP-1 is mirrored by enhanced clearance of such strains in animal models of endovascular infection; in contrast, in vitro resistance to tPMP-1 is correlated with an augmented survival advantage in the same animal models. The overall purposes of this proposal are: i) to define the mechanisms by which tPMP-1 executes its microbicidal effects, particularly focusing on intracellular targeting and activation of stress response systems; and ii) to delineate the mechanisms, genetic pathways and membrane biochemical adaptations by which the organism is able to successfully respond to exposures to tPMP-1 for survival. For these purposes, we will utilize a series of well-characterized and isogenic strain pairs of S. aureus (including site-directed plasmid mutants, as well as mutants with plasmid reporter fusions) that will enable us to define both the mechanisms of microbicidal action of tPMP-1, as well as the homeostatic adaptive pathways used by the organism to survive tPMP-1 exposures. Moreover, we will employ proteomics approaches to divulge novel genes and metabolic pathways triggered by tPMP-1 as part of either its microbicidal cascade, or as part of the organism's adaptive strategies. These studies will provide a solid foundation for the future design of unique platelet peptide congeners which are better able to target S. aureus strains for killing, as well as to circumvent innate homeostatic mechanisms used by the organism for survival.